Table 1.
Technique | SCRaMbLE | Mass mating/genome shuffling | Transcription machinery engineering/rewiring | Deletion library screening | Targeted deletion/overexpression/underexpression | Random mutagenesis/directed evolution |
---|---|---|---|---|---|---|
Strain prerequisites | Synthetic chromosomes and recombinase vectors | Natural isolates with desired properties | Efficient transformation into strain | Deletion library, efficient transformation into strain | — | — |
Prior knowledge | — | Phenotypic analysis of strains | Identification of transcriptional regulators/regulated promoters | — | Prior gene characterisation studies, knowledge of metabolic processes | — |
Preparation work | Transform pathway into one strain | Transform pathway into half of mating strains | Large-scale library construction | Transform pathway into many strains | Construction of TAR cassettes | Transform pathway into one strain |
Technical requirements | Basic strain cultivation | Strain mating/protoplast fusion, tetrad dissection | DNA assembly, library scale DNA transformation | Library scale DNA transformation | DNA assembly/gene editing tools | Strain cultivation, chemostat/tubidostat, mutagenesis |
Timescale to improved strain | 1–2 weeks | Months to years | Months to years | Weeks to months | Months | Months to years |
Strain diversity generated | Coding sequence deletions, inversions, duplications, translocations | Allele combinations, polyploidy | Novel regulation | Coding sequence deletions | Specific targeted changes | Point mutations |
Other factors | — | High prevalence of deleterious alleles, mitotic instability | High throughput screening often necessary | Higher numbers of combined knockouts exponentially increases transformation requirements | Understanding of biological mechanisms often not sufficient for effective improvement | High number of off-target effects, selective pressure often required |
Technical requirements do not include screening methods, which are common to all methods. Timescale to improved strain estimates length of time from introduction of a heterologous pathway to isolation and characterisation of a strain with an improved phenotype